Cell therapeutic agent for cancer treatment and combination therapy with same

ABSTRACT

The present disclosure relates to a method for preparing cells for cancer treatment and a kit for cancer treatment comprising cells prepared by the method. The preparation method of the present disclosure can provide F cells, which, in spite of having no difference in the proliferative capacity compared with mesenchymal stem cells expressing cytosine deaminase that are harvested and used immediately after the culture, exhibit a very excellent tumor suppressive effect through the treatment together with 5-FC and induce a remarkable synergistic effect exceeding an effect from combinative treatment with an existing anticancer drug in cases of a combinative treatment with another anticancer drug. Therefore, the present disclosure can be utilized for a kit for cancer treatment comprising such F cells, and thus can be favorably used to maximize the effect of existing cancer treatments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase under the provisions of 35U.S.C. §371 of International Patent Application No. PCT/KR2016/002188filed Mar. 4, 2016, which in turn claims priority of Korean PatentApplication No. 10-2015-0031751 filed Mar. 6, 2015. The disclosures ofsuch international patent application and Korean priority patentapplication are hereby incorporated herein by reference in theirrespective entireties, for all purposes.

TECHNICAL FIELD

The present disclosure relates to a method for preparing cells forcancer treatment, an adjuvant for anti-cancer comprising cells preparedby the same, a kit for cancer treatment, and a method for cancertreatment.

BACKGROUND ART

Millions of people around the world die from various types of cancerincluding bone cancer, bladder cancer, blood cancer (leukemia), braincancer, breast cancer, colorectal cancer, cervical cancer, esophagealcancer, bowel cancer, kidney cancer, lung cancer, liver cancer, oralcancer, nasal cancer, neural cancer, ovarian cancer, pancreatic cancer,skin cancer, stomach cancer, prostate cancer, neck cancer, uterinecancer, and vaginal cancer. Over the years, several methods includingradiation and chemotherapy have been used to treat cancer, but thenumber of cancer patients is still increasing. It has been widelyreported that radiation and chemotherapy may cause toxicity relateddiseases and even some patients die. In addition, in the case ofspecific cancer, there is a problem in that malignant cells remaindifficult to treat. As a result, it is required to extensively studyphysiology or phenotype of cancer cells to find a treatment method thatselectively kills the cancer cells without causing adverse effects onthe patient's healthy cells.

In particular, a brain tumor is a type of cancer that causes greatdamage to the brain and has a very low survival rate. Among conventionaltreatment methods for the brain tumor, extraction by surgical operationis the most effective treatment, but there are a number of cases in thatthe surgery is not possible depending on the type and location of thebrain tumor, and the risk of postoperative complications is very high atthe time of complete extraction. Further, since a brain-blood barrier(BBB) that inhibits drug penetration is present in the brain, in orderto treat brain tumor by chemotherapy using an anti-cancer agent, it isrequired to administer a high concentration of anti-cancer agent ascompared to other types of cancer, which causes serious side effects inother organs of the body. Therefore, there is a need for a noveladjuvant method, a therapeutic agent, and an adjuvant that are able tolower the dose of the anti-cancer agent by enhancing effects of theanti-cancer agent.

In addition, a gene therapy method is a method of directly introducing agene that inhibits proliferation of cancer cells into the cancer cells.For gene introduction, a virus is mainly used, but since the virusitself does not have an ability to move to cancer, it is required toinject the virus surgically. However, it is practically impossible toinject the viruses into every microscopic tumor or cancer cells, andthus, there is a limitation in targeting the cancer cells.

In this connection, Korean Patent Registration No. 10-1022401 is knownas a method for introducing a suicide gene into a mesenchymal stem cellto enhance targeting and treat cancer. However, KR 10-1022401 does notdisclose a combination therapy adjuvant for improving an effect of ananti-cancer agent by manipulating the mesenchymal stem cell in a specialenvironment and administering the mesenchymal stem cell in combinationwith another anti-cancer agent, and does not disclose a method forperiodic administration.

Further, recently, a number of studies on combination therapies such ascombination therapy of two or more kinds of anti-cancer agents have beenconducted. In this regard, not only a combination therapy of achemotherapeutic agent, but also a combination therapy of achemotherapeutic agent in combination with different kinds ofanti-cancer therapies, etc., has been recently reported. However, in thecase of combination therapy, there are many cases in which combinationadministration of each drug shows a simple additive effect of the samedegree of drug efficacy. Further, unpredictable side effects have beenreported, for example, an effect of the drugs is rather deteriorated byan interaction of the drugs, etc. Thus, studies on the combinationadministration of drugs are still continuing.

In connection with the combination administration in cancer, there isalso a study on the combination therapy of chemotherapeutic agents andcell therapeutic agents, such as mesenchymal stem cells. However, in thecombination therapy using the mesenchymal stem cells, researches aimingat administration of cells such as autologous stem cells for recovery ofcells damaged after anti-cancer therapy by chemotherapeutic agents havebeen intensively conducted, and there are not many attempts to utilizethe stem cells themselves as therapeutic agents for combination therapy.

Therefore, there is a need to develop a therapeutic agent and atreatment method capable of having high targeting so that cancer ortumor therapeutic agents are able to be accurately transferred to acancer tissue, being non-toxic so as not to affect parts other than thetumor, and being repeatedly administered until cancer is eliminatedbecause of no immunotoxicity. Further, there is a need and requirementfor a novel therapeutic method, therapeutic agent, therapeutic adjuvantand combination therapy that are able to improve or enhance the efficacyof existing anti-cancer agents.

DISCLOSURE Technical Problem

The present inventors studied a novel cancer therapeutic agent that iscapable of overcoming the limitations of the existing chemotherapy,found that cells prepared by introducing a cytosine deamine gene into amesenchymal stem cell to prepare mesenchymal stem cell/cytosinedeaminase (MSC/CD), followed by freezing and thawing, could exhibit veryexcellent tumor suppressive effect and survival rate improvement effectas compared to simply cultivated MSC/CD cells, and maximize effects ofcombination therapy with existing anti-cancer agents, and completed thepresent disclosure.

An object of the present disclosure is to provide a method for preparing“F cells” which are cells usable as an adjuvant for cancer treatment, akit for cancer treatment comprising F cells, a combined administrationagent with the F cells and a chemotherapeutic agent, and a combinedadministration method.

Technical Solution

In order to achieve the foregoing objects, the present disclosureprovides a method for preparing F cells comprising: 1) introducingcytosine deaminase (CD) into a mesenchymal stem cell (MSC) to preparemesenchymal stem cell/cytosine deaminase (MSC/CD); 2) freezing theprepared MSC/CD to prepare frozen MSC/CD; and 3) thawing and suspendingthe frozen MSC/CD to prepare F cells.

In addition, the present disclosure provides an adjuvant for anti-cancercomprising the F cells prepared by the method.

Further, the present disclosure provides a kit for cancer treatmentcomprising the F cells prepared by the method, and 5-fluorocytosine(5-FC).

In addition, the present disclosure provides a method for preventing ortreating cancer comprising: 1) introducing cytosine deaminase (CD) intoa mesenchymal stem cell (MSC) to prepare mesenchymal stem cell/cytosinedeaminase (MSC/CD); 2) freezing the prepared MSC/CD to prepare frozenMSC/CD; 3) thawing and suspending the frozen MSC/CD to prepare F cells;4) administering the F cells to a subject in need of treatment; and 5)administering 5-fluorocytosine (5-FC) to a subject in need of treatment.

Advantageous Effects

According to the preparation method of the present disclosure, it ispossible to provide the F cells, in spite of having no difference in theproliferative capacity compared with mesenchymal stem cells expressingcytosine deaminase, which are harvested and used immediately afterculturing, exhibit a very excellent tumor suppressive effect through thetreatment together with 5-FC, and induce a remarkable synergistic effectexceeding an effect from combination therapy with an existinganti-cancer agent, even in cases of a combination therapy with anotheranti-cancer agent. Therefore, the present disclosure may be utilized asa kit for cancer treatment comprising such F cells, and thus may befavorably used to maximize the effect of existing cancer treatments.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic diagram of a mesenchymal stem cell line (MSC/CD)that stably expresses cytosine deaminase, a suicide effect thereof, anda bystander effect.

FIG. 2 shows treatment process of MSC or MSC/CD with 5-FC in graph (A)and results of the suicide effect in graph (B) derived therefrom.

FIG. 3 shows treatment of MSC or MSC/CD with 5-FC in graph (A) andresults of the bystander effect in fluorescent images (B) and graph (C)derived therefrom.

FIG. 4 shows process of cell preparation and treatment of I cell, F cell(DMSO), and F cell (CS10) in graph (A) for comparing bystander effect onrespective cells and bystander effects in graph (B) on respective cells.

FIG. 5 shows a microscopic observation of a colony forming ability ofthe I cell and the F cell in images (A), and a graph for comparingquantitative analysis results using CFU-A (colony forming unit assay) ingraph (B).

FIG. 6 shows a process of treating the I-cell or the F-cell with 5-FC ina brain tumor model in graph (A), results of brain tumor changesfollowing the treatment observed under a dissecting microscope obtainedby observing brain tumor changes by the treatment in images (B), andtumor volume changes in graph (C).

FIG. 7 shows a graph for tumor volume changes according to the I cell orF cell treatment in graph (A) in subcutaneous tumor models in whichliver cancer cells are implanted subcutaneously, and representativetumor models showing an external size of the tumor in images (B).

FIG. 8 shows tumor volume changes according to the I cell or F celltreatment, in subcutaneous tumor models in which pancreatic cancer cellsare implanted subcutaneously in graph (A), in subcutaneous tumor modelsin which lung cancer cells are implanted subcutaneously in graph (B),and in subcutaneous tumor models in which colon cancer cells areimplanted subcutaneously in graph (C).

FIG. 9 shows F cells+5-FC+TMZ combination therapy in graph (A), thebystander effects derived therefrom in graphs (B) and (C), confirmationon results of cell death through fluorescence images of U87MG expressingGFP in images (D), and isobologram results of ICso values in graph (E).

FIG. 10 shows F cells+5-FC+anti-cancer agent combination therapy ingraph (A), isobologram results of ICso values of carmustine in graph(B), and isobologram results of ICso values of irinotecan in graph (C),respectively, in anti-cancer agent combination therapy.

FIG. 11 shows process of F cells+5-FC+temozolomide (TMZ) combinationtherapy in graph (A), the results of brain tumor changes following thetreatment observed under a dissecting microscope showing brain tumorchanges in images (B), tumor volume changes in graph (C), andsynergistic effects in improvement of survival rates in graph (D) by thecombination therapy.

FIG. 12 shows the analysis process on effects of the I cell or the Fcell+5-FC+temozolomide (TMZ) combination therapy in the subcutaneoustumor models in graph (A), tumor volume changes in graph (B), andsynergistic effects of the F cell+5-FC+temozolomide (TMZ) in analysis ofsurvival rates in graph (C).

BEST MODE

The present disclosure provides a method for preparing F cellsincluding: 1) introducing cytosine deaminase (CD) into a mesenchymalstem cell (MSC) to prepare mesenchymal stem cell/cytosine deaminase(MSC/CD); 2) freezing the prepared MSC/CD to prepare frozen MSC/CD; and3) thawing and suspending the frozen MSC/CD to prepare F cells.

According to the method for preparing F cells of the present disclosure,it is possible to provide the F cells, in spite of having no differencein the proliferative capacity compared with mesenchymal stem cellsexpressing cytosine deaminase, which are harvested and used immediatelyafter culturing I cells, exhibit a very excellent tumor suppressiveeffect when treated with 5-FC, and induce a remarkable synergisticeffect exceeding an effect from combination therapy with an existinganti-cancer agent, even in cases of a combination therapy with anotheranti-cancer agent.

The “MSC/CD” used herein refers to a mesenchymal stem cell prepared toexpress cytosine deaminase (CD) in the mesenchymal stem cell. Themesenchymal stem cell into which the cytosine deaminase is introducedrefers to a cell that moves to the vicinity of cancer cells with atropism towards the cancer cells, and continuously expresses cytosinedeaminase to induce suicide effect itself, and converts an non-toxicprodrug into an active anti-cancer agent to exhibit a bystander effectin which surrounding cancer cells are killed. The MSC/CD is advantageousin that since it undergo cell death, side effects that may be caused byundesired continuous effects may be reduced, and since it moves to thevicinity of cancer cells, infiltrative cancer cells may be killed,thereby acting as an effective anti-cancer agent. In particular, the “Fcell”, which is a cell prepared by freezing/thawing the MSC/CD, exhibitssynergistic effect in which an anti-cancer effect is significantlyincreased when used in combination with the existing anti-cancer agent,thereby being utilized as an adjuvant enhancing the effect of theanti-cancer agent.

The term “mesenchymal stem cell (MSC)” of the present disclosure is akind of stem cells that are able to be collected from bone marrow andumbilical cord blood, and is referred to a multipotent stromal cell thatis capable of proliferation, and in particular, that is capable of beingdifferentiated into various types of cells such as bone cells, cartilagecells, muscle cells, and fat cells, unlike hematopoietic stem cells.Therefore, the mesenchymal stem cell of the present disclosure maypreferably be the multipotent mesenchymal stem cell.

Term “cytosine deaminase” of the present disclosure is a suicide gene,which functions to convert a prodrug harmless to the human body into acytotoxic anticancer material, and specifically, a gene that converts5-fluorocytosine (5-FC) which is a prodrug of 5-fluorouracil (5-FU),into 5-FU.

The suicide gene may be introduced into the mesenchymal stem cellaccording to methods for intracellular introduction of genes known inthe art, such as a method of using a non-viral vector or a virus vectorincluding the same, or a physical method. Examples of such physicalmethods for gene transfer may include electroporation, hydroporation,injection, etc. Examples of gene transfer using non-viral vectors mayinclude methods of using cationic lipids, lipid polymers, ornanoparticles. The viral vector may include, without limitation, areplicable viral vector, a non-replicable viral vector, such as anadenoviral vector, an adeno-associated viral vector, a retroviralvector, herpes simplex virus, a hybrid adenoviral system, pox virus, alentivirus vector, and Epstein Barr virus.

For example, in an exemplary embodiment of the present disclosure, thesuicide gene may be introduced into the retrovirus vector to constructan expression vector, the vector may be transfected into packagingcells, the transfected packaging cells may be cultured and filtered toobtain a retrovirus solution, and this retrovirus solution may be usedto transfect the mesenchymal stem cell, thereby inserting the suicidegene into the mesenchymal stem cell. Next, the mesenchymal stem cellthat continuously expresses the suicide gene may be obtained using aselection marker included in the retrovirus vector.

In addition, since mesenchymal stem cell is known to have lowimmunorejection even during allotransplantation, it is possible to useeither cells isolated from others or cells collected from patients withcancer themselves, and to further minimize immunorejection amongindividuals through separation from bone marrow having similar HLA(human leukocyte antigen) types using a database of blood banks.

The MSC/CD of the present disclosure may be obtained by introducing asuicide gene into the isolated stem cell at first, followed byscreening, proliferation and differentiation under an appropriatecondition in a test tube, or may be obtained by sufficientlyproliferating the stem cell, introducing a suicide gene into the stemcell, followed by differentiation into the mesenchymal stem cell. TheMSC/CD is injected into a subject in need of treatment by the presenceof the suicide gene, and kills themselves after, for example, 2 to 30days, preferably 5 to 15 days, more preferably 5 to 10 days, but is notlimited thereto, from the injection, thereby reducing side effects thatmay be induced by continuous action.

Term “F cell (frozen cell)” of the present disclosure refers to a cellthat is not undergone an additional culturing step, free of culturing Fcells, after freezing and thawing the MSC/CD, which is the mesenchymalstem cell into which the cytosine deaminase (CD) is introduced, and isdefined as a cell being thawed and suspended, or suspended and washedwith a purpose of administration of cells to the subject to be treated.More specifically, the F cell refers to a cell obtained by introducingcytosine deaminase (CD) into the mesenchymal stem cell to prepare theMSC/CD, culturing, freezing the cell in a freezing medium, thawing thecell at room temperature immediately before use, followed by suspension,for example, in a Plasma Solution A, or the like, containing human serumalbumin, which is not limited thereto, and centrifugation. As a celldifferentiated from the F cell of the present disclosure, an immediatelyharvested cell (I cell)” may be exemplified, which is obtained byperforming the same freezing and thawing step on the MSC/CD, butmaximizing the concentration and activity through the subsequentculturing process, and is used by directly harvesting the cell in theculture.

The F cell, compared to the I cell, of the present disclosure ischaracterized in that the anti-cancer effect is increased withoutculturing step, which is performed generally to maximize activation andwithout increasing a colony forming ability. In addition, the F cell ofthe present disclosure may have an advantage of being immediatelyadministered to a patient in need of treatment together with theimprovement of the anti-cancer effect.

In the present disclosure, the cells in a cryopreserved state or frozenstate may be stored by using any means known in the art for maintainingthe cells to be in frozen state, such as a freezing preservation liquid,a freezing medium, etc. For example, the means may include mediaincluding freezing protectants such as dimethylsulfoxide (DMSO),dextran, human serum, human serum albumin, bovine serum, bovine serumalbumin, poly-1-lysine, polyvinylpyrrolidone, hydroxy-ethyl-starch,ethylene glycol, polyethylene glycol, glycerol, and percoll (forexample, cryostor CS2, cryostor CS10 commercially available from BioLifeSolutions Inc.), and specifically, cryostor CS2, cryostor CS10, and 2 to10% DMSO medium, and more specifically, 2, 5, and 10% DMSO media, etc.

The F cell of the present disclosure exhibits in vitro colony formingability similar to that of the I cell, and also effectively exhibits thebystander effect of the MSC/CD. Further, the F cell exhibits aninhibitory effect in vivo on a variety of tumors including brain tumorsand subcutaneous tumors twice or higher as compared to the I cell or thecontrol group, and exhibits a highly elevated anti-cancer effect in thecombination therapy with temozolomide (TMZ) which is the existinganti-cancer agent. In other words, even though the F cell has nodifference in the colony forming ability as compared to the I cell, theF cell exhibits a remarkable anti-cancer effect that are not able to beexpected in vitro and an effect as an anti-cancer agent adjuvant, ascompared to other I cells, due to the intrinsic activity of the F cellgiven according to the preparation method.

That is, the F cells of the present disclosure may be differentiatedfrom I cells that are conventionally used, by preparation methods inwhich cell culturing is not further performed after freezing.

Therefore, the present disclosure provides the method for preparing Fcells characterized in that the F cells are not subjected to cellculturing after freezing.

The F cell of the present disclosure is a cell for cancer treatment thatis usable for cancer treatment. The F cells may be used to treat,without limitation, all carcinomas capable of being extracted as cancerand carcinoma, and may be administered to a subject in need of treatmentbefore, simultaneously with, and after chemotherapy using administrationof anti-cancer agent.

The cancer may be, for example, at least one selected from the groupconsisting of squamous cell cancer (for example, squamous cell cancer ofepithelium), small cell lung cancer, non-small cell lung cancer, lungcancer, peritoneal cancer, colorectal cancer, biliary tumor,nasopharyngeal cancer, laryngeal cancer, bronchial cancer, oral cancer,osteosarcoma, gallbladder cancer, kidney cancer, leukemia, bladdercancer, melanoma, brain cancer, glioma, brain tumor, skin cancer,pancreatic cancer, breast cancer, liver cancer, bone cancer, esophagealcancer, colon cancer, gastric cancer, cervical cancer, prostate cancer,ovarian cancer, head and neck cancer, and rectal cancer, and morepreferably, brain tumor, pancreatic cancer, liver cancer, lung cancer,colon cancer, and skin cancer.

Further, the F cell of the present disclosure may also be an adjuvantfor anti-cancer. The adjuvant for anti-cancer refers to an adjuvant thatis capable of inducing faster action of a primary therapy and stronglyforming an anti-cancer action thereof by combination therapy with theprimary therapy, for example, cancer treatment by chemical therapy orsurgical operation. The adjuvant for anti-cancer may be used to enhancethe effect of anti-cancer agent, which is a main therapeutic agentprimarily used in chemotherapy using the anti-cancer agent, and may beused to maximize cancer treatment effect by treating residual cancercells after surgery.

The F cells may be present in a form of a composition for use in cancertherapy or for use in the adjuvant for anti-cancer, and such acomposition may include pharmaceutically acceptable excipients, carriersand diluents. A particularly preferred injection form is preferablyformulated into an injection form suitable for injection into a tissueor organ.

Accordingly, the present disclosure provides an adjuvant for anti-cancerincluding the F cells.

The F cell may be injected into the patient's body according to thephysician's prescription or by a method well known in the art, and asingle dose will be determined in consideration of various relatedfactors such as a disease to be treated, the severity of the disease,the route of administration, the body weight, age, and sex of thepatient, etc.

Further, the present disclosure provides a kit for cancer treatmentincluding the F cells, and 5-fluorocytosine (5-FC).

The kit for cancer treatment is prepared for the purpose of thecombination therapy of F cells and 5-fluorocytosine (5-FC), and means akit prepared so that the F cells are able to be injected before,simultaneously with, and after administration of 5-fluorocytosine (5-FC)which is a prodrug, to a subject in need of treatment.

The kit for cancer treatment may comprise a first compartment and asecond compartment, wherein the first compartment may comprise the Fcells for cancer treatment or the F cells acting as an adjuvant foranti-cancer, and the second compartment may include 5-fluorocytosine(5-FC). The kit may be contained in one container or in severaldifferent small containers with each divided dosage in order to improveconvenience and portability. Therefore, several containers may exist ineach container according to an arrangement method. The kit of thepresent disclosure may include, if necessary, equipment necessary foruse, instructions including descriptions of administration methods foreach component, etc.

In addition, a kit for cancer treatment of the present disclosure mayfurther include an anti-cancer agent. The anti-cancer agent may include,but is not limited to, anti-cancer agents known in the art, andpreferably may be at least one selected from the group consisting ofnitrogen mustard, imatinib, oxaliplatin, rituximab, elotinib,trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib,bevacizumab, cetuximab, viscum album, asparaginase, tretinoin,hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin,ibritumomab tiuxetan, heptaplatin, methylaminolevulinic acid, amsacrine,alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan,gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine,gimeracil, oteracil, azacytidine, cytarabine, fludarabine, enocitabine,decitabine, mercaptopurine, thioguanine, cladribine, carmofur,raltitrexed, docetaxel, paclitaxel, belotecan, topotecan, vinorelbine,etoposide, vincristine, vinblastine, tenifocide, idarubicin, epirubicin,mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin,pirarubicin, aclarubicin, pepromycin, temozolomide, busulfan,ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine,thiotepa, nimustine, chlorambucil, mitolactol, taxotere, gleevec, taxol,herceptin, tarceva, avastin, zoladex, adriamycin, irinotecan, 10058-F4,cisplatin, cyclophosphamid, nitrosourea-based anti-cancer agent,methotrexate, and doxorubicin. The nitrosourea includes carmustine,lomustine, and the like. The anti-cancer agent may be included in thethird compartment of the kit for cancer treatment.

In addition, the present disclosure provides a method for preventing ortreating cancer including: 1) introducing cytosine deaminase (CD) into amesenchymal stem cell (MSC) to prepare mesenchymal stem cell/cytosinedeaminase (MSC/CD); 2) freezing the prepared MSC/CD to prepare frozenMSC/CD; 3) thawing and suspending the frozen MSC/CD to prepare F cells;4) administering the F cells to a subject in need of treatment; and 5)administering 5-fluorocytosine (5-FC) to a subject in need of treatment.

The subject is preferably a mammal, including a human, and may includeall of a patient in need of cancer treatment, a patient undergoingcancer treatment, a patient experienced with cancer treatment, and apatient who need to be treated for cancer, and may also include apatient who underwent surgery to extract the cancer for cancertreatment.

Further, the present disclosure provides the method for preventing ortreating cancer, further including: 6) administering an anti-canceragent to a subject in need of treatment.

Further, the present disclosure provides a method for preventing ortreating cancer including: 4) administering an anti-cancer agent to asubject in need of treatment before, simultaneously with administrationof the F cells of step 4).

That is, the present disclosure provides the method for preventing ortreating cancer including both of simultaneous administration of the Fcells and the anti-cancer agent to a subject in need of treatment, andsequential administration, such as administration of the anti-canceragent after administration of the F cells, and administration of the Fcells after administration of the anti-cancer agent to a subject in needof treatment.

The anti-cancer agent may include, but is not limited to, anti-canceragents known in the art, and preferably may be at least one selectedfrom the group consisting of nitrogen mustard, imatinib, oxaliplatin,rituximab, elotinib, trastuzumab, gefitinib, bortezomib, sunitinib,carboplatin, sorafenib, bevacizumab, cetuximab, viscum album,asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine,gemtuzumab ozogamicin, ibritumomab tiuxetan, heptaplatin,methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine,alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine,pemetrexed, tegafur, capecitabine, gimeracil, oteracil, azacytidine,cytarabine, fludarabine, enocitabine, decitabine, mercaptopurine,thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel,belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine,tenifocide, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin,daunorubicin, dactinomycin, pirarubicin, aclarubicin, pepromycin,temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan,altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol,taxotere, gleevec, taxol, herceptin, tarceva, avastin, zoladex,adriamycin, irinotecan, 10058-F4, cisplatin, cyclophosphamid,nitrosourea-based anti-cancer agent, methotrexate, and doxorubicin. Thenitrosourea includes carmustine (BCNU), lomustine (CCNU), etc. Theanti-cancer agent may be appropriately and selectively prescribedaccording to a prescription dose of the anticancer agent which is widelyknown in the art, but may be prescribed at a dose smaller than anaverage prescription dose in consideration of the synergistic effectwith the F cells of the present disclosure.

The F cell of the present disclosure and 5-fluorocytosine may beadministered simultaneously or sequentially with time differences, whichmay be selected depending on appropriate time and period for conversionof 5-fluorocytosine to 5-fluorouracil by the F cell.

Therefore, the present disclosure provides the method for preventing ortreating cancer characterized in that the F cells of step 4) and the5-fluorocytosine of step 5) are administered simultaneously orsequentially.

Further, the anti-cancer agent used in combination with the5-fluorocytosine of the present disclosure may be administeredsimultaneously or sequentially with time differences, and may beselected according to appropriate time and period.

Therefore, the present disclosure provides the method for preventing ortreating cancer characterized in that the 5-fluorocytosine of step 5)and the anti-cancer agent of step 6) are administered simultaneously orsequentially.

The sequential administration in the present disclosure means thatmultiple components to be administered are administered to a subjectwith time differences, and the order of each of active components may beappropriately adjusted.

The F cells may be delivered in a pharmaceutically effective amount to atumor site of the subject, and the delivery is preferably performed byan injection form, but is not limited thereto. In particular, it is morepreferable that the injection is administered for mammals, preferably ahuman patient.

The F cells are the MSC/CD cells that are immediately used by freezing,thawing, or thawing and washing, and have an cytotoxic effect, which maybe killed in a subject body for a predetermined period of time by asuicide effect, preferably 5 to 15 days after injection. Thus, the Fcells may be repeatedly administered for continuous treatment. Further,the 5-FC, the anti-cancer agent may be repeatedly administered dependingon the therapeutic purpose and the condition of the patient. The cycleof the repeated administration of the F cells, 5-FC, and the anti-canceragent may be appropriately adjusted according to the therapeuticcondition of the patient, and the cycle may be preferably every 10 to 30days. For example, in consideration of the cell death of the MSC/CD, therepeated administration is preferable after at least 10 days, and therepeated administration every 30 days is also preferable consideringthat the cells are injected with a syringe.

Therefore, the present disclosure provides the method for preventing ortreating cancer characterized in that the administration of the F cellsof step 4) is repeated every 10 to 30 days.

Depending on the repetition of the F cell administration cycle, the5-fluorocytosine may also be repeatedly administered periodically, andoptionally, the anti-cancer agent for combined administration may alsobe repeatedly administered periodically.

In the method for preventing or treating cancer of the presentdisclosure, F cells may be used to treat, without limitation, allcarcinomas capable of being extracted as cancer and carcinoma, and maybe administered to a subject in need of treatment before, simultaneouslywith, and after chemotherapy using administration of anti-cancer agent.The cancer may be, for example, at least one selected from the groupconsisting of squamous cell cancer (for example, squamous cell cancer ofepithelium), small cell lung cancer, non-small cell lung cancer, lungcancer, peritoneal cancer, colorectal cancer, biliary tumor,nasopharyngeal cancer, laryngeal cancer, bronchial cancer, oral cancer,osteosarcoma, gallbladder cancer, kidney cancer, leukemia, bladdercancer, melanoma, brain cancer, glioma, brain tumor, skin cancer,pancreatic cancer, breast cancer, liver cancer, bone cancer, esophagealcancer, colon cancer, gastric cancer, cervical cancer, prostate cancer,ovarian cancer, head and neck cancer, and rectal cancer, and morepreferably, brain tumor, pancreatic cancer, liver cancer, lung cancer,colon cancer, and skin cancer.

Hereinafter, preferred Preparation Examples and Examples of the presentdisclosure will be described to assist in understanding the presentdisclosure. However, the following Preparation Examples and Examples areprovided only to more easily understand the present disclosure, andtherefore, the present disclosure is not limited thereto.

Preparation Example 1: Preparation of Mesenchymal Stem Cell ExpressingCytosine Deaminase (CD)

1.1 Isolation and Culture of Mesenchymal Stem Cell

4 ml of human bone marrow donated after the IRB examination of AjouUniversity Medical Center was overlaid on 4 ml of HISTOPAQUE 1077(Sigma-Aldrich) in a sterilized 15 ml test tube, and centrifuged at400×g for 30 minutes at room temperature using a centrifuge. Aftercentrifugation, 0.5 ml of the intermediate buffy coat was carefullycollected using a Pasteur pipette, and transferred to a test tubecontaining 10 ml of sterilized phosphate buffer solution (pH 7.4) Aftercentrifugation at 250×g for 10 minutes, the supernatant was discarded,and 10 ml of phosphate buffer solution was added and gently suspended,followed by centrifugation at 250×g for 10 minutes. This procedure wasrepeated twice, and the final precipitate was added to a DMEM medium(Gibco) supplemented with 10% FBS (Hyclone) and dispensed in a 100 mmanimal cell culture dish to obtain 1×10⁹ cells. The culture containerwas placed in an incubator and cultured at 37° C. for 4 hours whilesupplying 5% carbon dioxide and 95% air. Subsequently, in order toremove cells that did not stick to the bottom of the culture container,the supernatant was removed. A fresh medium was added thereto, andcultured in an incubator.

The isolated mesenchymal stem cells were maintained in a CO₂ incubatorat 37° C., and cultured in a mesenchymal stem cell culture medium [(10%FBS (Gibco)+10 ng/ml bFGF (Sigma-Aldrich)+1% penicillin/streptomycin(Gibco)+89% DMEM (Gibco)]. The cells were cultured while replacing themedium every two days. When the cells reached about 80% in the culturecontainer, the cells were isolated using 0.25% trypsin/0.1 mM EDTA(Gibco), diluted so as to satisfy about 800 to 1,1000 cells/cm², or 1:10to 1:20 with the medium, and subcultured in a new culture container.Some of the cells were stored frozen by a freezing medium supplementedwith 2-10% DMSO (Sigma-Aldrich, 2, 5, 10%).

1.2 Preparation of Retrovirus Including Cytosine Deaminase (CD)

A retrovirus expressing cytosine deaminase (CD), a suicide gene, wasconstructed. Detailed description thereof is as follows:

DNA was isolated from E. coli K-12 MG1655 (Korea Research Institute ofBioscience and Biotechnology), and then PCR was performed underconditions of 5 minutes at 94° C., 30 seconds at 94° C., 40 seconds at60° C., 1 minute at 72° C., 27 cycles; 7 minutes at 72° C. using primersCD-F(5′-GAA TTC AGG CTA GCA ATG TCT CGA ATA ACG CTT TAC AAA C-3′) andCD-R (5′-GGATTC TCT AGC TGG CAG ACA GCC GC-3′). The PCR product wasusing a pGEM-T Easy vector cloning kit (Promega), and then a PGEM-CDvector containing the CD gene was constructed through blue-white colonyscreening using X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside,Sigma-Aldrich) and IPTG (Isopropyl 13-D-1-thiogalactopyranoside,Sigma-Aldrich). It was confirmed that the base sequence of the obtainedCD gene was identical to that of gi: 298594 of GeneBank through sequenceanalysis. The prepared pGEM-CD plasmid and pcDNA3.1 (Clontech) weredigested with EcoRI and Notl restriction enzymes, respectively, and theCD gene isolated from the plasmid pGEM-CD and the cut plasmid pcDNA3.1were ligated with T4 DNA ligase. Then, the obtained product was used totransform E. coli DH5a, a competent cell, and cultured on an LB platecontaining 50 μg/ml of ampicillin and selected to obtain a plasmidpcDNA3.1-CD. This plasmid was digested with BamH I restriction enzymeand introduced into a retroviral vector capable of producing retrovirus.The obtained plasmid was isolated by a CsCl-ultra high-speed centrifuge,and transfected into FLYRD18 cell, which is a retrovirus packaging cellline, by calcium phosphate precipitation (Jordan, Nucleic Acid Research,24, 596-601 (1996)). Then, the cells were cultured at 37° C. in anincubator while supplying 5% carbon dioxide and 95% air. After 48 hours,only the culture solution was obtained and filtered through a 0.45 IAfilter membrane to obtain a retrovirus solution. The retrovirus solutionwas dispensed and used while storing at −70° C.

1.3 Preparation of Mesenchymal Stem Cell into which CD Gene isIntroduced

The CD gene was introduced into the mesenchymal stem cell isolated andcultured in the description of 1.1, using the CD-containing retrovirusprepared in the description of 1.2. More specifically, the mesenchymalstem cell was cultured to reach about 70% in a 100 mm culture dish.Then, 3 ml of the retrovirus solution, 3 ml of the fresh mesenchymalstem cell culture medium, and 4 μg/ml of polybrene (Sigma-Aldrich) wereadded and cultured for 8 hours. Then, the virus solution was removed, 10ml of mesenchymal stem cell culture medium was added and cultured for 16hours, and then infected again with retrovirus. After this procedure wasperformed 1 to 3 times, the mesenchymal stem cell was finally taken offwith trypsin and subcultured by 1:20 dilution with the medium. In thesubculture, puromycin (Sigma-Aldrich) was added to the medium to aconcentration of 2 μg/ml. The cells were screened for 2 weeks so thatonly cells infected with retrovirus could survive. Finally, themesenchymal stem cell line (hereinafter, referred to as MSC/CD)continuously expressing CD, which is a suicide gene, was constructed.

Example 1. Suicide Effect and Bystander Effect of MSC/CD

1.1 Confirmation of Suicide Effect

The cytosine deaminase (CD) is a suicide gene. The MSC/CD, which is amesenchymal stem cell expressing the cytosine deaminase (CD), mayconvert 5-fluorocytosine (5-FC) which is a prodrug into 5-fluorouracil(5-FU) and may have a suicide effect (the cells kill themselves) and abystander effect (peripheral cells are killed) as shown in FIG. 1.Therefore, the suicide effect and the bystander effect of the MSC/CDprepared in the description of 1.3 were confirmed. First, 10,000 MSC/CDor mesenchymal stem cells (MSCs) were cultured in a 12-well plate. Fromthe next day, the cells were treated with the prodrug 5-FC at aconcentration of 0 to 1,000 μM, and replaced with a new mediumcontaining the drug once every two days. The medium was changed to acell culture medium containing 0.5 mg/ml of MTT(3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide,Sigma-Aldrich) capable of measuring living cells on the 6^(th) day aftertreatment with 5-FC, and reacted for 2 hours at 37° C. Then, the MTTsolution was removed. 500 μl of DMSO was added to each well to perform acolor reaction. Then, the lysates were transferred to a 96-well plateand absorbance was measured at 540 nm using an ELISA reader (E-max,Molecular device). A schematic diagram of the experimental method andthe results thereof are shown in FIG. 2, in graphs (A) and (B) thereof.

As shown in FIG. 2, in graphs (A) and (B) thereof, it was confirmed thatthe MSC/CD had a suicidal effect in which the living cells were reducedas the concentration of the prodrug 5-FC was increased, and ICsoinducing 50% cell death was 60.4 μM. In the case of mesenchymal stemcell, which is the control group, it was confirmed that even if theconcentration of 5-FC was increased, the cells did not kill themselves,and thus, the cytototoxic effect was not observed.

1.2 Bystander Effect

10,000 GFP-expressing U87MG (Korean Cell Line Bank KCLBNo. 30014) gliomacells and 10,000 MSC/CD or mesenchymal stem cells (MSCs) were culturedon a 12-well plate. From the next day, the cells were treated with theprodrug 5-FC at a concentration of 0 to 1,000 μM, and replaced with anew medium containing the drug once every two days, and after 6 days,the cells were obtained. Such an experimental method is briefly shown inFIG. 3, in graph (A) thereof.

Then, fluorescence images of U87MG expressing GFP remaining in the wholewell were taken using an dissecting fluorescence microscope (Olympus).After the fluorescence images were taken, 200 μl of 1× passive lysisbuffer (Promega) was added, and the cell lysate was placed on ice for 10minutes and transferred to an E-tube and centrifuged at 12,000 rpm for 5minutes, and the supernatant was transferred to a new container. 100 μlof the supernatant was transferred to a black 96-well plate, and thedegree of fluorescence was measured under conditions of excitation 488nm and emission 530 nm using a fluorimeter (GEMINI EM, moleculardevice). The results are shown in FIG. 3, in fluorescence images (B) andbystander effects graph (C) thereof.

As shown in FIG. 3, in fluorescence images (B) and bystander effectsgraph (C) thereof, U87MG cultured with MSC/CD showed a decrease in thenumber of cells expressing fluorescence and a remarkable decrease influorescence intensity as the concentration of 5-FC was increased, butU87MG cultured with the mesenchymal stem cells in which the CD was notexpressed did not show a significant difference in the number of cellsexpressing fluorescence and fluorescence intensity according to theconcentration of 5-FC. Herein, IC₅₀ inducing 50% cell death was 50.48μM. The results indicated that the MSC/CD, which is the mesenchymal stemcell into which the CD was introduced, showed not only the suicideeffect but also the bystander effect in which the peripheral cells werekilled together.

Preparation Example 2. Preparation of “I Cell” and “F Cell”

In the case of a cell therapy agent, the effect may vary depending onthe change in the cell state under various conditions. Therefore, inorder to confirm whether the cell ability was changed according to thestate of MSC/CD, cells in a thawed state immediately after freezing(hereinafter, referred to as F cells (frozen cells)), and cells in aharvested state immediately from the cells during the culturing(hereinafter, referred to as I cells (immediately harvested cells)) wereprepared.

In order to prepare the F cells and the I cells, respectively, theMSC/CD was cultured, and then 2×10⁶ cells were dispensed in 1 ml offreezing medium containing 2 to 10% (2, 5, 10%) DMSO or 2×10⁶ cells weredispensed in 1 ml of Cryostor CS10 (BioLife Solutions). The cells werefrozen and stored in a liquid nitrogen container. First, in order toobtain “I cells” of MSC/CD, cells of passage 5 were dissolved at 37° C.,and placed in 9 ml of culture medium. Then, the cells were centrifugedat 500 g for 5 minutes to remove the supernatant, and the precipitatedcells were suspended in the culture solution, and dispensed in a 150 mmculture container. The next day, 1.5×10⁵ cells were transferred to the150 mm culture container, and replaced with new medium once every twodays. After 6 days, the cells were collected to prepare “I cells”corresponding to passage 6.

In order to obtain the “F cells”, the MSC/CD frozen and stored in thepassage 6 at −130° C. or below was dissolved at 37° C. and suspended ina Plasma Solution A (CJ HealthCare Corp.) containing 9 ml of human serumalbumin, followed by centrifugation at 500×g for 5 minutes, or suspendedin physiological saline containing a final 7.5% dextran-40 (Daihan PharmCo., Ltd.) and 5% human serum albumin (Green Cross Corp.), followed bycentrifugation at 800×g for 10 minutes. Then, the supernatant wasremoved, and the precipitated cells were suspended in the culture mediumand used. In other words, the F cells are cells that were not subjectedto the culturing step after freezing and thawing. The I cells and the Fcells were washed twice, respectively, before transplantation into thebrain.

Example 1. Bystander Effect and Colony Formation of F Cells

1.1 Bystander Effect of F Cells

Experiments were performed to determine whether the F cells couldnormally exhibit the bystander effect of MSC/CD and whether thebystander effect appeared effectively regardless of the preparationmethod such as the type of medium. 10,000 I cells prepared by the methodof Preparation Example 2, 10,000 F cells prepared with 10% DMSO freezingmedium (DMSO), and 10,000 F cells prepared with Cryostor CS10 (CS10)were washed with a Plasma Solution A, and were co-cultured in 12-wellplates together with glioma U87/GFP cells in which GFP gene wastransduced into U87MG (KCLB No. 30014) donated from Korean Cell LineBank so as to express fluorescence. From the next day, the cells weretreated with the prodrug 5-FC at a concentration of 0 to 1,000 μM, andreplaced with a new medium containing the drug once every two days, andafter 6 days, the cells were obtained. 200 μl of 1× passive lysis buffer(Promega) was added, and the cells were placed on ice for 10 minutes.The cell lysate was transferred to an E-tube and centrifuged at 12,000rpm for 5 minutes, and the supernatant was transferred to a newcontainer. 100 μl of the supernatant was transferred to a black 96-wellplate in which light is blocked, and the degree of fluorescence wasmeasured under conditions of excitation 488 nm and emission 530 nm usinga fluorimeter (GEMINI EM, molecular device). A schematic diagram of theexperimental method and the bystander effect are shown in FIG. 4, ingraphs (A) and (B) thereof.

As shown in FIG. 4, in graphs (A) and (B), the IC₅₀ values indicatingconcentration at which 50% of apoptosis is caused, was 60.4 μM for Icells, 56.2 μM for F cells prepared with 10% DMSO freezing medium(DMSO), and 55.4 μM for F cells prepared with Cryostor CS10 (CS10).These results indicated that the F cell was a cell that couldeffectively exhibit the bystander effect as compared to the I cell, andthus it was confirmed that the F cell exhibited the bystander effectmore excellently as compared to the I cell regardless of the preparationmethod using the DMSO medium or the CS10 medium.

Therefore, hereinafter, the F cells prepared with the 10% DMSO freezingmedium were used among the F cells prepared in Preparation Example 2.

1.2. Colony Forming Effect of F Cells

A colony forming unit assay was performed to confirm whether the F cellsprepared by freezing and thawing as in Preparation Example 2 exhibited adifference in proliferative capacity as compared to the I cells. The Icells prepared as in Preparation Example 2 and corresponding the samepassage and the F cells prepared with 10% DMSO freezing medium werecounted with Countess (Invitrogen), and then 100 cells were placed in a100 mm culture container, respectively, and cultured while replacing themedium with a new medium once every two days. After 2 weeks, the cellswere fixed with 10% formalin for 10 minutes, washed with phosphatebuffer solution (Gibco), stained with a crystal violet solution(Sigma-Aldrich) for 10 minutes, and washed three times with water toobtain images by Versa doc (BioRad). The number of colonies formed bygrowing one cell for 2 weeks was visually counted, and shown in FIG. 5,in image (A) and graph (B) thereof.

As shown in FIG. 5, in image (A) and graph (B) thereof, it was confirmedthat the I and F cells had no difference in the colony forming ability.These results indicated that the increased bystander effect of the Fcells as compared to that of the I cells was not caused by an increasein the number of cells such as an increase in colony formation, and thatthe F cells themselves exhibited more excellent bystander effect ascompared to that of the I cells.

Example 2. Anti-Cancer Effect of F Cell

2.1. Anti-Cancer Effect of F Cell on Brain Tumor

In order to confirm the anti-cancer effect of the F cells on braintumor, an orthotopic glioma model was constructed. First, 7-week-oldimmunodeficient nude mice (central experimental animals) wereanesthetized and their mouths and ears were fixed in a sterotaxic frame.The head to be incised was sterilized with 70% ethanol, and the crown ofhead was vertically incised to about 0.5 cm. 3×10⁵/3 μl of U87MG gliomacells expressing LacZ were transplanted at a rate of 0.3 μl/min at thebrain coordinates of AP=+0.5 mm, ML=−1.8 mm, and DV=−3 mm, therebyconstructing a brain tumor orthotopic glioma model. On the 3rd day aftertransplantation of U87MG glioma cells expressing LacZ, the F cellsprepared in Preparation Example 2 were suspended in a Plasma Solution A,followed by centrifugation at 500×g for 5 minutes. The supernatant wasdiscarded, and the washing procedure was repeated once or twice. Then,the cell suspension was prepared to have a concentration of 3×10⁵/6 μl.The cell suspension was transplanted at a rate of 0.3 μl/min at thebrain coordinates of AP=+0.5 mm, ML=−1.8 mm, and DV=−3 mm. From the nextday after the cell transplantation, 5-FC (15 mg/ml physiological saline)was intraperitoneally injected at a dose of 500 mg/kg for one week. Tocompare the anti-cancer effect of the F cells, the I cells prepared inPreparation Example 2 were transplanted in the same manner, and 5-FC (15mg/ml physiological saline) was intraperitoneally injected at a dose of500 mg/kg for one week from the next day after the cell transplantation.

On the 28^(th) day after glioma cell transplantation, the animals wereanesthetized, and perfused with physiological saline by inserting aninjection needle into the left ventricle of the animals, and then,perfused with 10% formalin solution. The brain was extracted and fixedon a mouse brain matrix (Stoelting). Then, the brain was cuthorizontally at intervals of 1 mm, and put in the same fixing fluid for30 minutes, thereby fixing the brain slice. The brain slice was washedthree times with phosphate buffered saline, immersed in phosphatebuffered saline containing 20 mg/ml of X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, Koma Biotech), 5 mMpotassium ferrocyanide/ferricyanide (Sigma-Aldrich), 2 mM MgCl₂, and0.02% NP40, and reacted at 37° C. for 16 hours. An image of the braintumor was obtained through an anatomic microscope. A size of the tumorwas calculated using the image j (NIH) program and the followingEquation, and the results are shown in FIG. 6, in graph (A), images (B),and graph (C) thereof.

${{Tumor}\mspace{14mu} {volume}\mspace{11mu} \left( {mm}^{3} \right)} = {\sum{\frac{{Number}\mspace{14mu} {of}\mspace{14mu} {pixels}\mspace{14mu} {of}\mspace{14mu} {tumor}\mspace{14mu} {area}}{{Number}\mspace{14mu} {of}\mspace{14mu} {pixels}\mspace{14mu} {per}\mspace{14mu} {unit}\mspace{14mu} {area}} \times 0.5\mspace{14mu} {mm}}}$

As shown in FIG. 6, in images (B) thereof, in the F cell transplantationgroup, the volume of the brain tumor was remarkably decreased, and moreexcellent effect was exhibited as compared to the I cell. The computedmean size of the tumor was 91.3 mm³ in the vehicle control group, 1.5mm³ in the I cell transplantation comparison group, but 0.6 mm³ in the Fcell transplantation animal group. That is, according to the in vivoresults, it was confirmed that the F cell had a tumor-reducing effecttwice more than that of the I cell among the MSC/CD, and the F cellcould be a more effective anti-cancer agent.

2.2. Anti-Cancer Effect of F Cell on Liver Cancer

1×10⁵ Huh7 liver cancer cells (KCLB No. 60104) were suspended in 100 μlof a phosphate buffer solution containing 20% Matrigel (BD), andtransplanted subcutaneously in 7-week-old immunodeficient nude mice(Balb/c nude). After 14 days from transplantation, when the tumor sizereached 10 to 100 mm³, the I cells and the F cells prepared inPreparation Example 2 were injected. More specifically, the I cells andF cells prepared in Preparation Example 2 were suspended in thephosphate buffer solution, followed by centrifugation at 500×g for 5minutes. This procedure was repeated twice, and then the I cells or theF cells were prepared to have concentration of 1×10⁶/100 μl in thephosphate buffer solution and injected directly into the tumor using asyringe. From the next day after the I cell and F cell injection, 5-FC(15 mg/ml physiological saline) was intraperitoneally injected at a doseof 500 mg/kg for one week. The size of the tumor was measured once ortwice weekly using a digital caliper, and the tumor volume wascalculated using the following Equation, and the results are shown inFIG. 7, in graph (A) and animal images (B) thereof.

Subcutaneous tumor volume (mm³)=Width (mm)×Length (mm)×Height (mm)÷6

[For measurement of tumor volume, see Cancer Chemother. Pharmacol.(1989) 24: 148-154]

As shown in graph (A) of FIG. 7, the tumor volume was more effectivelydecreased in the experimental group treated with the F cell, confirmingthat the F cell had more excellent anti-cancer effect than the I cell.As shown in animal images (B) of FIG. 7, the increased anti-cancereffect was visually confirmed in the subcutaneous tumor modeltransplanted with the liver cancer cells, and thus, it was confirmedthat the therapeutic effect of F cells was excellent (*, p<0.05 uponcomparison with the PBS group).

2.3. Anti-Cancer Effect of F Cell on Pancreatic Cancer

BxPC3 pancreatic cancer cells (ATCC No. CRL-1687™) had a rapid growthrate and severe ulceration, and thus, 1×10⁶ BxPC3 pancreatic cancercells, the I cells, and the F cells were injected simultaneously. Morespecifically, the I cells and F cells prepared in Preparation Example 2were suspended in the phosphate buffer solution, followed bycentrifugation at 500×g for 5 minutes. This procedure was repeatedtwice, and then the I cells or the F cells were prepared to haveconcentration of 1×10⁶/100 μl in the phosphate buffer solution. 1×10⁶ Icells or F cells were suspended in 100 μl of a phosphate buffer solutioncontaining 20% Matrigel (BD), and simultaneously transplantedsubcutaneously in 7-week-old immunodeficient nude mice (Balb/c nude).From the next day after the cell injection, 5-FC (15 mg/ml physiologicalsaline) was intraperitoneally injected at a dose of 500 mg/kg for oneweek.

The tumor volume was measured in the same manner as in Example 2.2, andthe measured tumor volume change was shown in FIG. 8, in graph (A)thereof.

As shown in graph (A) of FIG. 8, the tumor volume was more effectivelydecreased in the experimental group treated with the F cell, confirmingthat the F cell had more excellent anti-cancer effect than the I cell,and had excellent therapeutic effect in the pancreatic cancersubcutaneous tumor model (*, p<0.05 upon comparison with the PBS group).

2.4. Anti-Cancer Effect of F Cell on Lung Cancer

5×10⁶ A549 lung cancer cells (ATCC No. CCL-185™) were suspended in 100μl of a phosphate buffer solution containing 20% Matrigel (BD), andtransplanted subcutaneously in 7-week-old immunodeficient nude mice(Balb/c nude). After 8 days from transplantation, when the tumor sizereached 20 to 50 mm³, the I cells and the F cells prepared inPreparation Example 2 were injected. More specifically, the I cells andF cells prepared in Preparation Example 2 were suspended in thephosphate buffer solution, followed by centrifugation at 500×g for 5minutes.

This procedure was repeated twice, and then the I cells or the F cellswere prepared to have concentration of 1×10⁶/100 μl in the phosphatebuffer solution and injected directly into the tumor using a syringe.From the next day after the I cell and F cell injection, 5-FC (15 mg/mlphysiological saline) was intraperitoneally injected at a dose of 500mg/kg for one week. The tumor volume was measured in the same manner asin Example 2.2, and the measured tumor volume change was shown in FIG.8, in graph (B) thereof.

As shown in graph (B) of FIG. 8, the tumor volume was more effectivelydecreased in the experimental group treated with the F cell, confirmingthat the F cell had more excellent anti-cancer effect than the I cell,and had excellent therapeutic effect in the lung cancer subcutaneoustumor model (*, p<0.05 upon comparison with the PBS group).

2.5. Anti-Cancer Effect of F Cell on Colon Cancer

5×10⁶ HT29 colon cancer cells (ATCC No. HTB-38™) were suspended in 100μl of a phosphate buffer solution containing 20% Matrigel (BD), andtransplanted subcutaneously in 7-week-old immunodeficient nude mice(Balb/c nude). After the transplantation, when the tumor size reached 40to 100 mm³, the I cells and the F cells prepared in Preparation Example2 were injected. More specifically, the I cells and F cells prepared inPreparation Example 2 were suspended in the phosphate buffer solution,followed by centrifugation at 500×g for 5 minutes.

This procedure was repeated twice, and then the I cells or the F cellswere prepared to have concentration of 1×10⁶/100 μl in the phosphatebuffer solution and injected directly into the tumor using a syringe.From the next day after the I cell and F cell injection, 5-FC (15 mg/mlphysiological saline) was intraperitoneally injected at a dose of 500mg/kg for one week. The tumor volume was measured in the same manner asin Example 2.2, and the measured tumor volume change was shown in FIG.8, in graph (C) thereof.

As shown in graph (C) of FIG. 8, the tumor volume was more effectivelydecreased in the experimental group treated with the F cell, confirmingthat the F cell had more excellent anti-cancer effect than the I cell,and had excellent therapeutic effect in the colon cancer subcutaneoustumor model (*, p<0.05 upon comparison with the PBS group, and #, p<0.05upon comparison with the I cell group).

From the above results, it was confirmed that the F cell had anexcellent anti-cancer effect in various types of cancer such as braintumor, liver cancer, lung cancer, colon cancer, and exhibited a moreexcellent anti-cancer effect in all types of cancer as compared to the Icell.

Example 3. Synergistic Effect of Combination Therapy of F Cells (InVitro)

3.1 In Vitro Combination Therapy with Temozolomide

10,000 GFP-expressing U87MG (Korean Cell Line Bank KCLBNo. 30014) gliomacells and 10,000 F cells or mesenchymal stem cells (MSCs) were culturedin a 12-well plate. The next day, the cells were treated withtemozolomide (TMZ) at a concentration of 0 to 1000 μM and the prodrug5-FC at a concentration of 0 to 1,000 μM, and replaced with a new mediumcontaining the drug once every two days, and after 6 days, the cellswere obtained. A schematic diagram of this experiment is shown in graph(A) of FIG. 9. Fluorescence images of U87MG expressing GFP remaining inthe whole well were taken using a dissecting fluorescence microscope(Olympus), and cell death was confirmed. The results are shown in thefluorescence images (D) of FIG. 9.

As shown in the fluorescence images (D) of FIG. 9, it was confirmed thatthe fluorescence expression was decreased according to an increase inthe temozolomide treatment concentration and an increase in the 5-FCtreatment concentration, and the bystander effect of the F cell waselevated by the combined administration with temozolomide.

After the fluorescence images were taken, 200 μl of 1× passive lysisbuffer (Promega) was added, and the cells were placed on ice for 10minutes. The cell lysate was transferred to an E-tube and centrifuged at12,000 rpm for 5 minutes, and the supernatant was transferred to a newcontainer. 100 μl of the supernatant was transferred to a black 96-wellplate, and the intensity of fluorescence was measured under conditionsof excitation 488 nm and emission 530 nm using a fluorimeter (GEMINI EM,molecular device) (graphs (B) and (C) of FIG. 9). The actual ICso valuesobtained by treating the two types of anti-cancer agents together wereexpressed as isobologram. The solid line connecting the ICso values whenthe anti-cancer agent was treated alone in the isobologram is atheoretical additive line, which means that when the values obtained bysimultaneously treating two anti-cancer agents are on the solid line,there is simply an additive effect. On the other hand, it means thatwhen the value is lower than the solid line, there is a synergisticeffect, and conversely, when the value is above the solid line, there isan antagonistic effect. The results are shown in FIG. 9, in graph (E)thereof.

As shown in graph (E) of FIG. 9, all of the marked points when theMSC/CD was treated with 5-FC and temozolomide in combination werepresent at the lower side as compared to the ICso values when theanti-cancer agent was treated alone, which was confirmed that there wasa synergistic effect which is more excellent than the additive effectobtained by simple treatment with the two anti-cancer agents together.

3.2 In Vitro Combination Therapy with Carmustine (BCNU)

10,000 GFP-expressing U87MG glioma cells, i.e., U87/GFP (Korean CellLine Bank KCLBNo. 30014) and 10,000 F cells (MSC/CD) were cultured in a12-well plate. From the next day, the cells were treated with Carmustine(BCNU, Sigma) at a concentration of 0 to 300 μM and the prodrug 5-FC ata concentration of 0 to 300 μM, and cultured for 6 days while replacingwith a new medium containing the drug once every two days. On the 7thday, the culture solution was removed, 200 μl of 1× passive lysis buffer(Promega) was then added to each well, and each well was allowed tostand at 4° C. for 10 minutes. The cell lysate was collected andcentrifuged at 12,000 rpm for 5 minutes to obtain a supernatant. 100 μlof the supernatant was transferred to a black 96-well plate in whichlight is blocked, and the intensity of fluorescence was measured underconditions of excitation 488 nm and emission 530 nm using a fluorimeter(GEMINI EM, molecular device). A schematic diagram of this experiment isshown in graph (A) of FIG. 10.

The actual ICso values obtained by treatment with the two types ofanti-cancer agents together were expressed as isobologram, and theresults were shown in FIG. 10, in graph (B) thereof.

As shown in graph (B) of FIG. 10, all of the marked points when U87/GFPwas treated with [F cell (MSC/CD)+5-FC] and carmustine in combinationwere present at the lower side of the solid line as compared to the ICsovalues when the anti-cancer agent was treated alone, which was confirmedthat there was a synergistic effect which is more excellent than theadditive effect obtained by simple treatment with the two anti-canceragents together.

3.3 In Vitro Combination Therapy with Irinotecan

10,000 U87/GFP and 10,000 F cells (MSC/CD) were cultured in a 12-wellplate. From the next day, the cells were treated with Irinotecan (Sigma)at a concentration of 0 to 30 μM and the prodrug 5-FC at a concentrationof 0 to 300 μM, and cultured for 6 days while replacing with a newmedium containing the drug once every two days. On the 7th day, theculture solution was removed, 200 μl of 1× passive lysis buffer was thenadded to each well, and each well was allowed to stand at 4° C. for 10minutes. The cell lysate was collected and centrifuged at 12,000 rpm for5 minutes to obtain supernatant. 100 μl of the supernatant wastransferred to a black 96-well plate in which light is blocked, and theintensity of fluorescence was measured under conditions of excitation488 nm and emission 530 nm using a fluorimeter (GEMINI EM). The actualICso values obtained by treatment with the two types of anti-canceragents together were expressed as isobologram, and the results wereshown in graph (C) of FIG. 10.

As shown in graph (C) of FIG. 10, the ICso values obtained by treatmentwith irinotecan alone in the isobologram was 3.2 μM and the ICso valuesobtained by treatment with 5-FC was 73.6 μM, and these ICso values wereconnected to obtain a theoretical additive line representing a simpleadditive effect. The ICso values obtained when U87/GFP was treated with[F cell (MSC/CD)+5-FC] and irinotecan in combination were present at thelower side of the solid line as compared to the ICso values when theanti-cancer agent was treated alone, which was confirmed that there wasa synergistic effect which is more excellent than the effect obtained bysimple treatment with the two anti-cancer agents together.

Since all of temozolomide, carmustine, and irinotecan are known asanti-cancer agents that act on proliferating cells, if they affect thesurvival of not only U87/GFP cells but also proliferating F cells, theanti-cancer effect of [F cell (MSC/CD)+5-FC] might be reduced. However,contrary to the expectation, it was confirmed from graph (E) of FIG. 9that the synergistic effect could be obtained which was rather elevatedas compared to the administration of the anti-cancer agent alone or incombination, when [F cell (MSC/CD)+5-FC] was used in combination withtemozolomide. That is, the synergistic effect is an unexpected effectthat is not capable of being expected in the existing combinationtherapies.

3.4 In Vivo Combination Therapy with Temozolomide

The synergistic effects confirmed in the above-descriptions of 3.1 to3.3 were further confirmed and verified in vivo. On the 6th days aftertransplantation of U87MG glioma cells expressing LacZ, the F cellsprepared in Preparation Example 2 were suspended in a Plasma Solution A,followed by centrifugation at 500×g for 5 minutes. The supernatant wasdiscarded, and the washing procedure was repeated twice. Then, the cellsuspension was prepared to have a concentration of 3×10⁵/6 μl. The cellsuspension was transplanted at a rate of 0.3 μl/min at the braincoordinates of AP=+0.5 mm, ML=−1.8 mm, and DV=−3 mm. From the next dayafter the cell transplantation, 5-FC (15 mg/ml physiological saline) wasintraperitoneally injected at a dose of 500 mg/kg for one week. After 4days, temozolomide (1 mg/ml in DMSO:physiological saline=1:1) wasintraperitoneally injected at a dose of 5 mg/kg for 5 days. On 28th day,brain tissues were obtained from 8 animals in each animal group andX-gal staining was performed in the same manner as in Example 2 tomeasure the size of brain tumor. The experimental procedure is shown ingraph (A) of FIG. 11, and the changes in measured brain tumor size areshown in images (B) and graph (C) of FIG. 11.

As shown in images (B) and graph (C) of FIG. 11, the average size ofvehicle control tumors was 59.8 mm³, whereas the average size of theanimal group into which the F cells were transplanted was 11.8 mm³, theaverage size of the animal group treated with temozolomide alone was 9.9mm³, and the average size of the animal group into which the Fcells+temozolomide were transplanted was 2.7 mm³. The animal group intowhich the F cells and temozolomide combination therapy were transplantedhad the most reduced average size of the tumor, and showed the mostexcellent anti-cancer effect.

In addition, the survival rate according to the treatment of brain tumorwas measured up to 90 days, and the results were compared in eachexperimental group and showed in graph (D) of FIG. 11.

As shown in graph (D) of FIG. 11, the median survival in the controlgroup was 32 days, but prolonged to 42 days in the F cell treatmentgroup and 42 days in the temozolomide treatment group. In particular,the median survival was prolonged to 70 days in the group with the Fcell and temozolomide combination therapy, and a remarkable improvementin the survival rate was observed even in comparison with the grouptreated with the anti-cancer agent alone as well as the control group.

This suggests that the combination therapy with F cell showed aremarkably excellent effect even in the combination therapy with theexisting anti-cancer agents, and thus, the combination therapy with Fcell is able to be utilized as a remedy for improving the existingchemotherapy.

Example 4. Subcutaneous Tumor Treatment Effect of F Cells

To determine whether the F-cell could exhibit the anti-cancer effect intumors other than brain tumor, U87MG glioma cells were transplanted intosubcutaneous tissue to construct a non-brain cancer model. 1×10⁶ U87MGglioma cells were suspended in 100 μl of a phosphate buffer solutioncontaining 20% Matrigel (BD), and transplanted subcutaneously in7-week-old immunodeficient nude mice. The size of the tumor was measuredtwice weekly using a digital caliper, and the tumor volume wascalculated using the following Equation.

Subcutaneous tumor volume (mm³)=Width (mm)×Length (mm)×Height (mm)÷6

When the size of the tumor reached 30 mm³ or more on 20^(th) day afterthe U87MG glioma cells were transplanted into the subcutaneous tissue,the I cells and F cells prepared in Preparation Example 2 were suspendedin a Plasma Solution A, and centrifuged at 500×g for 5 minutes. Thisprocedure was repeated twice, and then the I cells or the F cells wereprepared to have concentration of 1×10⁶/100 μl in the Plasma Solution Aand injected directly into the tumor using a syringe. From the next dayafter the I cell and F cell injection, 5-FC (15 mg/ml physiologicalsaline) was intraperitoneally injected at a dose of 500 mg/kg for oneweek. After one week from the administration of 5-FC was discontinued,temozolomide (Sigma-Aldrich) was intraperitoneally injected at a dose of5 mg/kg for 5 days. Such an experimental method is briefly shown ingraph (A) of FIG. 12. The measured tumor volume change is shown in graph(B) of FIG. 12. The Kaplan Meier survival graph obtained by measuringthe lifespan of nude mice in the subcutaneous tumor model is shown ingraph (C) of FIG. 12.

As shown in graph (B) of FIG. 12, it was confirmed that the tumor volumewas more effectively decreased in the experimental group treated with Fcells until the 11^(th) day before the administration of temozolomide,and the F cells showed more excellent anti-cancer effect than the Icells. Further, on the 21^(st) day after the administration oftemozolomide, the tumor volume inhibitory effect in the F cell treatmentgroup was confirmed to be very remarkable as compared to thenon-treatment group (the control group) and the I cell treatment group.In addition, as shown in graph (C) of FIG. 12, the median survival ofthe control group was 23 days, and the median survival of the I celltreatment group was 30 days, whereas the F cell treatment group showedthe median survival of 58 days, and thus, the life extension effect wasalso the greatest in the animal group treated with the F cells.Therefore, it may be appreciated that the F cells have excellenttherapeutic effect in the subcutaneous tumor model, and in particular,exhibit a synergistic therapeutic effect through combination therapywith temozolomide, which demonstrates superiority of the F cellcombination therapy in other organs, which is difficult to be expectedfrom in vitro experiment alone.

1. A method for preparing F cells comprising: 1) introducing cytosinedeaminase (CD) into a mesenchymal stem cell (MSC) to prepare mesenchymalstem cell/cytosine deaminase (MSC/CD); 2) freezing the prepared MSC/CDto prepare frozen MSC/CD; and 3) thawing and suspending the frozenMSC/CD to prepare F cells.
 2. The method according to claim 1, whereinthe F cell(s) is not subjected to cell culturing after freezing.
 3. Themethod according to claim 1, wherein the F cell(s) is used for cancertreatment.
 4. The method according to claim 3, wherein the cancer is atleast one selected from the group consisting of squamous cell cancer,small cell lung cancer, non-small cell lung cancer, lung cancer,peritoneal cancer, colorectal cancer, biliary tumor, nasopharyngealcancer, laryngeal cancer, bronchial cancer, oral cancer, osteosarcoma,gallbladder cancer, kidney cancer, leukemia, bladder cancer, melanoma,brain cancer, glioma, brain tumor, skin cancer, pancreatic cancer,breast cancer, liver cancer, bone cancer, esophageal cancer, coloncancer, gastric cancer, cervical cancer, prostate cancer, ovariancancer, head and neck cancer, and rectal cancer.
 5. The method accordingto claim 1, wherein the F cell(s) is an adjuvant for anti-cancer.
 6. Anadjuvant for anti-cancer comprising the F cells prepared by the methodaccording to claim
 1. 7. A kit for cancer treatment comprising the Fcells prepared by the method according to claim 1, and 5-fluorocytosine(5-FC).
 8. The kit according to claim 7, further comprising ananti-cancer agent.
 9. The kit according to claim 8, wherein theanti-cancer agent is at least one selected from the group consisting ofnitrogen mustard, imatinib, oxaliplatin, rituximab, elotinib,trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib,bevacizumab, cetuximab, viscum album, asparaginase, tretinoin,hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin,ibritumomab tiuxetan, heptaplatin, methylaminolevulinic acid, amsacrine,alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan,gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine,gimeracil, oteracil, azacytidine, cytarabine, fludarabine, enocitabine,decitabine, mercaptopurine, thioguanine, cladribine, carmofur,raltitrexed, docetaxel, paclitaxel, belotecan, topotecan, vinorelbine,etoposide, vincristine, vinblastine, tenifocide, idarubicin, epirubicin,mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin,pirarubicin, aclarubicin, pepromycin, temozolomide, busulfan,ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine,thiotepa, nimustine, chlorambucil, mitolactol, taxotere, gleevec, taxol,herceptin, tarceva, avastin, zoladex, adriamycin, irinotecan, 10058-F4,cisplatin, cyclophosphamid, nitrosourea-based anti-cancer agent,methotrexate, carmustine (BCNU), lomustine (CCNU), and doxorubicin. 10.A method for preventing or treating cancer comprising: 1) introducingcytosine deaminase (CD) into a mesenchymal stem cell (MSC) to preparemesenchymal stem cell/cytosine deaminase (MSC/CD); 2) freezing theprepared MSC/CD to prepare frozen MSC/CD; 3) thawing and suspending thefrozen MSC/CD to prepare F cells; 4) administering the F cells to asubject in need of treatment; and 5) administering 5-fluorocytosine(5-FC) to a subject in need of treatment.
 11. The method according toclaim 10, further comprising 6) administering an anti-cancer agent to asubject in need of treatment.
 12. The method according to claim 10,further comprising administering an anti-cancer agent to a subject inneed of treatment before administration of the F cells according to step4) or simultaneously with administration of the F cells according tostep 4).
 13. The method according to claim 11, wherein the anti-canceragent is at least one selected from the group consisting of nitrogenmustard, imatinib, oxaliplatin, rituximab, elotinib, trastuzumab,gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab,cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide,dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tiuxetan,heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab,procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine,doxifluridine, pemetrexed, tegafur, capecitabine, gimeracil, oteracil,azacytidine, cytarabine, fludarabine, enocitabine, decitabine,mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed,docetaxel, paclitaxel, belotecan, topotecan, vinorelbine, etoposide,vincristine, vinblastine, tenifocide, idarubicin, epirubicin,mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin,pirarubicin, aclarubicin, pepromycin, temozolomide, busulfan,ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine,thiotepa, nimustine, chlorambucil, mitolactol, taxotere, gleevec, taxol,herceptin, tarceva, avastin, zoladex, adriamycin, irinotecan, 10058-F4,cisplatin, cyclophosphamid, nitrosourea-based anti-cancer agent,methotrexate, carmustine (BCNU), lomustine (CCNU), and doxorubicin. 14.The method according to claim 10, wherein the F cells of step 4) and the5-fluorocytosine of step 5) are administered simultaneously orsequentially.
 15. The method according to claim 11, wherein the5-fluorocytosine of step 5) and the anti-cancer agent of step 6) areadministered simultaneously or sequentially.
 16. The method according toclaim 11, wherein the administration of the F cells according to step 4)is repeated with a cycle of 10 to 30 days.
 17. The method according toclaim 10, wherein the cancer is at least one selected from the groupconsisting of squamous cell cancer, small cell lung cancer, non-smallcell lung cancer, lung cancer, peritoneal cancer, colorectal cancer,biliary tumor, nasopharyngeal cancer, laryngeal cancer, bronchialcancer, oral cancer, osteosarcoma, gallbladder cancer, kidney cancer,leukemia, bladder cancer, melanoma, brain cancer, glioma, brain tumor,skin cancer, pancreatic cancer, breast cancer, liver cancer, bonecancer, esophageal cancer, colon cancer, gastric cancer, cervicalcancer, prostate cancer, ovarian cancer, head and neck cancer, andrectal cancer.